B-1 cells are murine B cells that are readily distinguishable from conventional B cells (B-2 cells), that preferentially localize in the peritonel cavity, are part of innate immunity, secrete natural antibodies in a T cell independent manner, and play important roles in the first line of defense against bacterial or viral infections, sepsis and atherosclerosis. Despite the importance of B-1 cells in host defense and homeostasis, their developmental origin is not clear. CD5+B-1a cells residing in the peritoneal cavity can be reconstituted by transplantation of fetal liver (FL) progenitors, but not by adult bone marrow (BM) hematopoietic stem cells (HSCs). Thus B-1a cells are considered to be of fetal origin and the mechanisms that maintain B-1a cells in adult life without being replenished by adult HSCs have yet to be elucidated. The overall goal of the project is to confirm the contribution of HSC-independent embryonic B-1 progenitor cells into postnatal B-1 lineages and define the mechanisms that maintain B-1 cells throughout adult life. We have reported that B-1 cells originate from hemogenic endothelial cells (HECs) in the yolk sac (YS) prior to HSC emergence in the mouse embryo. We now also report that B-1 progenitor cells are present in a HSC-deficient mouse model. Thus, we hypothesize that YS-derived (HSC independent) B-1a cells are maintained throughout perinatal and adult life. In order to prove this hypothesis, we will utilize a lineage tracing mouse model where one can mark HECs and their progeny at specific embryonic time point before HSC emergence by tamoxifen induction. We anticipate the ability to define the contribution of the embryonic-derived cells to adult B-1 cell pools in Aim 1. Mature B-1a cells display self-replenishing ability that is detectable by secondary transplantation. The molecular mechanisms that sustain this self-replenishing activity in B-1a cells are unknown. Bmi1 is a polycomb group protein that is critical for self-renewal of HSCs. We report that the self- replenishing capacity of Bmi1-/- B-1a cells is impaired; raising the hypothesis that Bmi1 plays an important role in B-1a cell self-renewal. We will examine the cell autonomous role of Bmi1 in B-1a cell self- renewal/maintenance and determine its molecular mechanisms in Aim 2. Finally, in Aim 3, we will capitalize on our recent identification of murine embryonic stem (ES) cell differentiation into B-1 cells to examine human induced pluripotent stem (iPS) cell differentiation for evidence of B cell generation. We anticipate that the B cells generated from the human iPS cells will display functional features of murine B-1 cells and provide a novel means to generate human B-1 cells. Our results will provide new insights into the origin and mechanisms of maintenance of murine B-1 cells and will unfold a novel method to generate human B-1 cells as a potential cell therapy.